PubMed Health⌕ Search

Biomedical subjects

H Ischiropoulos

Publications and source records attributed to H Ischiropoulos.

At least 19 recordsLinked to original sources
PubMed2001

Induction of alpha-synuclein aggregation by intracellular nitrative insult.

Brain lesions containing filamentous and aggregated alpha-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant alpha-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of alpha-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of alpha-synuclein. Light microscopy using antibodies against alpha-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. alpha-Synuclein aggregates were observed in 20-30% of cells expressing wild-type or A53T mutant alpha-synuclein and in 5% of cells expressing A30P mutant alpha-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing beta-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing alpha-synuclein. However, alpha-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote alpha-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of alpha-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of alpha-synuclein, indicating that lysosomes are involved in degradation of alpha-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha-synuclein aggregates.

Ammonium Chloride↗
PubMed2001

Nitration and inactivation of tyrosine hydroxylase by peroxynitrite.

Tyrosine hydroxylase (TH) is modified by nitration after exposure of mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine. The temporal association of tyrosine nitration with inactivation of TH activity in vitro suggests that this covalent post-translational modification is responsible for the in vivo loss of TH function (Ara, J., Przedborski, S., Naini, A. B., Jackson-Lewis, V., Trifiletti, R. R., Horwitz, J., and Ischiropoulos, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7659-7663). Recent data showed that cysteine oxidation rather than tyrosine nitration is responsible for TH inactivation after peroxynitrite exposure in vitro (Kuhn, D. M., Aretha, C. W., and Geddes, T. J. (1999) J. Neurosci. 19, 10289-10294). However, re-examination of the reaction of peroxynitrite with purified TH failed to produce cysteine oxidation but resulted in a concentration-dependent increase in tyrosine nitration and inactivation. Cysteine oxidation is only observed after partial unfolding of the protein. Tyrosine residue 423 and to lesser extent tyrosine residues 428 and 432 are modified by nitration. Mutation of Tyr(423) to Phe resulted in decreased nitration as compared with wild type protein without loss of activity. Stopped-flow experiments reveal a second order rate constant of (3.8 +/- 0.9) x 10(3) m(-1) s(-1) at pH 7.4 and 25 degrees C for the reaction of peroxynitrite with TH. Collectively, the data indicate that peroxynitrite reacts with the metal center of the protein and results primarily in the nitration of tyrosine residue 423, which is responsible for the inactivation of TH.

Base Sequence↗
PubMed2001

Dynamic regulation of metabolism and respiration by endogenously produced nitric oxide protects against oxidative stress.

One of the many biological functions of nitric oxide is the ability to protect cells from oxidative stress. To investigate the potential contribution of low steady state levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against H(2)O(2), spontaneously transformed human ECV304 cells, which normally do not express eNOS, were stably transfected with a green fluorescent-tagged eNOS cDNA. The eNOS-transfected cells were found to be resistant to injury and delayed death following a 2-h exposure to H(2)O(2) (50-150 microM). Inhibition of nitric oxide synthesis abolished the protective effect against H(2)O(2) exposure. The ability of nitric oxide to protect cells depended on the presence of respiring mitochondria as ECV304+eNOS cells with diminished mitochondria respiration (rho(-)) are injured to the same extent as nontransfected ECV304 cells and recovery of mitochondrial respiration restores the ability of nitric oxide to protect against H(2)O(2)-induced death. Nitric oxide also found to have a profound effect in cell metabolism, because ECV304+eNOS cells had lower steady state levels of ATP and higher utilization of glucose via the glycolytic pathway than ECV304 cells. However, the protective effect of nitric oxide against H(2)O(2) exposure is not reproduced in ECV304 cells after treatment with azide and oligomycin suggesting that the dynamic regulation of respiration by nitric oxide represent a critical and unrecognized primary line of defense against oxidative stress.

Adenosine Triphosphate↗
PubMed2001

SIN-1-induced DNA damage in isolated human peripheral blood lymphocytes as assessed by single cell gel electrophoresis (comet assay).

Human lymphocytes were exposed to increasing concentrations of SIN-1, which generates superoxide and nitric oxide, and the formation of single-strand breaks (SSB) in individual cells was determined by the single-cell gel electrophoresis assay (comet assay). A dose- and time-dependent increase in SSB formation was observed rapidly after the addition of SIN-1 (0.1-15 mM). Exposure of the cells to SIN-1 (5 mM) in the presence of excess of superoxide dismutase (0.375 mM) increased the formation of SSB significantly, whereas 1000 U/ml catalase significantly decreased the quantity of SSB. The simultaneous presence of both superoxide dismutase and catalase before the addition of SIN-1 brought the level of SSB to that of the untreated cells. Moreover, pretreatment of the cells with the intracellular Ca(2+)-chelator BAPTA/AM inhibited SIN-1-induced DNA damage, indicating the involvement of intracellular Ca(2+) changes in this process. On the other hand, pretreatment of the same cells with ascorbate or dehydroascorbate did not offer any significant protection in this system. The data suggest that H2O2-induced changes in Ca(2+) homeostasis are the predominant pathway for the induction of SSB in human lymphocytes exposed to oxidants.

Ascorbic Acid↗
PubMed2001

Nitrated and oxidized plasma proteins in smokers and lung cancer patients.

Cigarette smoking is a cause of lung cancer and other respiratory diseases. Oxidants either present in cigarette smoke and/or formed in the lung of smokers may trigger oxidative and nitrative damage to DNA and cellular components, contributing to carcinogenesis. We have used immunodot and Western blot analyses to measure nitrated (nitrotyrosine-containing) and oxidized (carbonyl-containing) proteins in plasma samples collected from 52 lung cancer patients and 43 control subjects (heavy and light smokers, nonsmokers with or without exposure to environmental tobacco smoke). The levels of nitrated proteins were significantly higher in lung cancer patients than in controls (P = 0.003). On the other hand, the levels of oxidized proteins were significantly higher in smokers than in nonsmokers (P < 0.001). Western-blot analyses showed the presence of two to five nitrated proteins and one oxidized protein. Using immunoprecipitation and Western-blot analyses with eight different antibodies against human plasma proteins, we identified fibrinogen, transferrin, plasminogen, and ceruloplasmin as nitrated proteins and fibrinogen as the only oxidized protein present in human plasma of lung cancer patients and smokers. Our results indicate that cigarette smoking increases oxidative stress and that during lung cancer development, formation of reactive nitrogen species results in nitration and oxidation of plasma proteins.

Aged↗
PubMed2001

Nitric oxide chemistry and cellular signaling.

Nitric oxide (NO) has been shown to mediate a number of different physiological functions within every major organ system. This wide variety of functional roles is made all the more remarkable when one considers that NO is a simple diatomic molecule. However, despite the simplicity of the molecule, NO possesses a wide range of chemical reactivity and multiple potential reactive targets. It is the variability of NO reactivity, which leads to its capability to control such a vast range of biological functions. In essence the functionality of NO is controlled by its chemical reactivity. In order to understand this possibility further it is necessary to consider the biologically relevant reactions of nitric oxide.

Animals↗
PubMed2001

Oxidative post-translational modifications of alpha-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease.

Structural and functional alterations of alpha-synuclein is a presumed culprit in the demise of dopaminergic neurons in Parkinson's disease (PD). Alpha-synuclein mutations are found in familial but not in sporadic PD, raising the hypothesis that effects similar to those of familial PD-linked alpha-synuclein mutations may be achieved by oxidative post-translational modifications. Here, we show that wild-type alpha-synuclein is a selective target for nitration following peroxynitrite exposure of stably transfected HEK293 cells. Nitration of alpha-synuclein also occurs in the mouse striatum and ventral midbrain following administration of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Conversely, beta-synuclein and synaptophysin were not nitrated in MPTP-intoxicated mice. Our data demonstrate that alpha-synuclein is a target for tyrosine nitration, which, by disrupting its biophysical properties, may be relevant to the putative role of alpha-synuclein in the neurodegeneration associated with MPTP toxicity and with PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2001

Tyrosine nitration: localisation, quantification, consequences for protein function and signal transduction.

The nitration of free tyrosine or protein tyrosine residues generates 3-nitrotyrosine the detection of which has been utilised as a footprint for the in vivo formation of peroxynitrite and other reactive nitrogen species. The detection of 3-nitrotyrosine by analytical and immunological techniques has established that tyrosine nitration occurs under physiological conditions and levels increase in most disease states. This review provides an updated, comprehensive and detailed summary of the tissue, cellular and specific protein localisation of 3-nitrotyrosine and its quantification. The potential consequences of nitration to protein function and the pathogenesis of disease are also examined together with the possible effects of protein nitration on signal transduction pathways and on the metabolism of proteins.

Animals↗
PubMed2001

Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation.

An elevation in inorganic phosphate (P(i)) concentration activates epiphyseal chondrocyte apoptosis. To determine the mechanism of apoptosis, tibial chondrocytes were treated with P(i), and nitrate/nitrite (NO/NO) levels were determined. P(i) induced a threefold increase in the NO/NO concentration; inhibitors of nitric oxide (NO) synthase activity and P(i) transport significantly reduced NO/NO levels and prevented cell death. Furthermore, a dose-dependent increase in cell death was observed after exposure of chondrocytes to S-nitrosoglutathione. P(i) increased caspase 3 activity 2.7-fold. Both caspase 1 and caspase 3 inhibitors protected chondrocytes from P(i)-induced apoptosis. P(i) caused a significant decrease in the mitochondrial membrane potential, while NO synthase inhibitors maintained mitochondrial function. While P(i) caused thiol depletion, inhibition of P(i) uptake or NO generation served to maintain glutathione levels. The results suggest that NO serves to mediate key metabolic events linked to P(i)-dependent chondrocyte apoptosis.

Animals↗
PubMed2000

Oxidative damage linked to neurodegeneration by selective alpha-synuclein nitration in synucleinopathy lesions.

Aggregated alpha-synuclein proteins form brain lesions that are hallmarks of neurodegenerative synucleinopathies, and oxidative stress has been implicated in the pathogenesis of some of these disorders. Using antibodies to specific nitrated tyrosine residues in alpha-synuclein, we demonstrate extensive and widespread accumulations of nitrated alpha-synuclein in the signature inclusions of Parkinson's disease, dementia with Lewy bodies, the Lewy body variant of Alzheimer's disease, and multiple system atrophy brains. We also show that nitrated alpha-synuclein is present in the major filamentous building blocks of these inclusions, as well as in the insoluble fractions of affected brain regions of synucleinopathies. The selective and specific nitration of alpha-synuclein in these disorders provides evidence to directly link oxidative and nitrative damage to the onset and progression of neurodegenerative synucleinopathies.

Alzheimer Disease↗
PubMed2000

Proteolytic degradation of tyrosine nitrated proteins.

Tyrosine nitration is a covalent posttranslational protein modification that has been detected under several pathological conditions. This study reports that nitrated proteins are degraded by chymotrypsin and that protein nitration enhances susceptibility to degradation by the proteasome. Chymotrypsin cleaved the peptide bond between nitrated-tyrosine 108 and serine 109 in bovine Cu,Zn superoxide dismutase. However, the rate of chymotryptic cleavage of nitrated peptides was considerably slower than control. In contrast, nitrated bovine Cu,Zn superoxide dismutase was degraded at a rate 1. 8-fold faster than that of control by a gradient-purified 20S/26S proteasome fraction from bovine retina. Exposure of PC12 cells to a nitrating agent resulted in the nitration of tyrosine hydroxylase and a 58 +/- 12.5% decline in the steady-state levels of the protein 4 h after nitration. The steady-state levels of tyrosine hydroxylase were restored by selective inhibition of the proteasome activity with lactacystin. These data indicate that nitration of tyrosine residue(s) in proteins is sufficient to induce an accelerated degradation of the modified proteins by the proteasome and that the proteasome may be critical for the removal of nitrated proteins in vivo.

Animals↗
PubMed2000

Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro.

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox(-/)-, iNOS(-/)-, and doubly immunodeficient phox(-/)-iNOS(-/)- mice. The respiratory burst and NO radical (NO.) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase-dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O(2).(-)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S. typhimurium. Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon gamma appears to augment antibacterial activity predominantly by enhancing NO. production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

Animals↗
PubMed2000

Cytochrome c nitration by peroxynitrite.

Peroxynitrite (ONOO(-)), the product of superoxide (O(2)) and nitric oxide (.NO) reaction, inhibits mitochondrial respiration and can stimulate apoptosis. Cytochrome c, a mediator of these two aspects of mitochondrial function, thus represents an important potential target of ONOO(-) during conditions involving accelerated rates of oxygen radical and.NO generation. Horse heart cytochrome c(3+) was nitrated by ONOO(-), as indicated by spectral changes, Western blot analysis, and mass spectrometry. A dose-dependent loss of cytochrome c(3+) 695 nm absorption occurred, inferring that nitration of a critical heme-vicinal tyrosine (Tyr-67) promoted a conformational change, displacing the Met-80 heme ligand. Nitration was confirmed by cross-reactivity with a specific antibody against 3-nitrotyrosine and by increased molecular mass compatible with the addition of a nitro-(-NO(2)) group. Mass analysis of tryptic digests indicated the preferential nitration of Tyr-67 among the four conserved tyrosine residues in cytochrome c. Cytochrome c(3+) was more extensively nitrated than cytochrome c(2+) because of the preferential oxidation of the reduced heme by ONOO(-). Similar protein nitration patterns were obtained by ONOO(-) reaction in the presence of carbon dioxide, whereupon secondary nitrating species arise from the decomposition of the nitroso-peroxocarboxylate (ONOOCO(2)(-)) intermediate. Peroxynitrite-nitrated cytochrome c displayed significant changes in redox properties, including (a) increased peroxidatic activity, (b) resistance to reduction by ascorbate, and (c) impaired support of state 4-dependent respiration in intact rat heart mitochondria. These results indicate that cytochrome c nitration may represent both oxidative and signaling events occurring during .NO- and ONOO(-)-mediated cell injury.

Animals↗
PubMed2000

Dityrosine cross-linking promotes formation of stable alpha -synuclein polymers. Implication of nitrative and oxidative stress in the pathogenesis of neurodegenerative synucleinopathies.

Intracellular proteinaceous aggregates are hallmarks of many common neurodegenerative disorders, and recent studies have shown that alpha-synuclein is a major component of several pathological intracellular inclusions, including Lewy bodies in Parkinson's disease (PD) and glial cell inclusions in multiple system atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into filamentous inclusions remain unknown. Since oxidative and nitrative stresses are potential pathogenic mediators of PD and other neurodegenerative diseases, we asked if oxidative and/or nitrative events alter alpha-synuclein and induce it to aggregate. Here we show that exposure of human recombinant alpha-synuclein to nitrating agents (peroxynitrite/CO(2) or myeloperoxidase/H(2)O(2)/nitrite) induces formation of nitrated alpha-synuclein oligomers that are highly stabilized due to covalent cross-linking via the oxidation of tyrosine to form o,o'-dityrosine. We also demonstrate that oxidation and nitration of pre-assembled alpha-synuclein filaments stabilize these filaments to withstand denaturing conditions and enhance formation of SDS-insoluble, heat-stable high molecular mass aggregates. Thus, these data suggest that oxidative and nitrative stresses are involved in mechanisms underlying the pathogenesis of Lewy bodies and glial cell inclusions in PD and multiple system atrophy, respectively, as well as alpha-synuclein pathologies in other synucleinopathies.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

Chaperone-like activity of synucleins.

Synucleins are a family of small proteins that are predominantly expressed in neurons. The functions of the synucleins are not entirely understood, but they have been implicated in the pathogenesis of several neurodegenerative diseases. Our data show that alpha-, beta- or gamma-synuclein suppresses the aggregation of thermally denatured alcohol dehydrogenase and chemically denatured insulin. The A53T but not the A30P mutant alpha-synuclein was able to inhibit the aggregation of insulin and the chaperone-like activity of alpha-synuclein was lost upon removal of its C-terminal residues 98-140. These results demonstrate that synucleins with the exception of the A30P mutant possess chaperone-like activity.

Alcohol Dehydrogenase↗
PubMed2000

Oxygen-insensitive nitroreductases of Escherichia coli do not reduce 3-nitrotyrosine.

The oxygen-insensitive nitroreductases nfsA and nfsB are known to reduce para-nitrated aromatic compounds. We tested the hypothesis that these nitroreductases are capable of reducing 3-nitrotyrosine in proteins and peptides, as well as in free amino acids using wild-type and nfsA nfsB mutant strains of Escherichia coli. E. coli homogenates were incubated with nitrated proteins and the level of 3-nitrotyrosine immunoreactivity was assayed by Western blotting. Assay conditions that allow the nitroreductases to rapidly reduce nitrofurantoin did not result in the modification of 3-nitrotyrosine in protein, peptide, or free amino acid. Stimulation of nfsA nfsB activity with paraquat had no effect on 3-nitrotyrosine reduction. Nonlethal exposure of E. coli to peroxynitrite/CO(2) resulted in the reproducible nitration of tyrosine residues in endogenous proteins. The degree of 3-nitrotyrosine immunoreactivity over the 2-h postexposure period did not differ between mutant and wild-type strains. These results indicate that the nfsA and nfsB enzymes do not reduce 3-nitrotyrosine.

Bacterial Proteins↗